Narayan Mahesh, Welker Ervin, Scheraga Harold A
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 2003 Jun 17;42(23):6947-55. doi: 10.1021/bi030054w.
A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-Pt(en)(2)Cl(2) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.
本文采用一种最近开发的方法来表征核糖核酸酶A的一些折叠中间体和氧化折叠过程。该方法基于反式-Pt(en)(2)Cl(2)能够比通过重排或还原重新排列二硫键更快地氧化半胱氨酸残基以形成二硫键的能力。这种方法的变体使我们能够解决三个问题。(i) 如何阐明在折叠初始阶段存在或形成的残余结构和/或构象有序性的性质。本文表明，与在强变性条件下（4-6 M盐酸胍）相比，核糖核酸酶A未折叠还原形式直接形成天然二硫键组的倾向增加了10倍。因此，核糖核酸酶A未折叠还原形式不是比盐酸胍变性状态下更紧凑形式的统计卷曲；相反，有人认为还原的核糖核酸酶A对天然拓扑结构有一点偏向。(ii) 展示了根据二硫键配对对氧化折叠中间体进行结构表征；具体而言，对核糖核酸酶A的二硫键组合中含天然二硫键分子的百分比进行了下限估计。(iii) 先前已表明结构化中间物种在确定蛋白质氧化折叠途径中的关键作用。在此，我们证明这种方法可以揭示蛋白质氧化折叠中结构化中间体的存在。
